1. Field of Invention
This invention relates to microcarrier beads for use in bioreactors and methods for growing cells, specifically anchorage dependent cells, upon porous structures. Further, this invention relates to methods of creating scaffolds for artificial tissues and also the scaffolds and tissues made by these methods.
2. Description of Related Art
Anchorage dependent cells are difficult to culture to commercial yields because they cannot grow in suspension. Mammalian cells, most of which are anchorage dependent, require the presence of oxygen, but they are highly sensitive to the shear and bubbles created by the fluid motion and sparging required for creating a high dissolved oxygen count.
The ability to grow mammalian cells is important for academia and industry to produce biological agents and biomaterials such as, for example, proteins, hormones, vaccines, antibodies, antibiotics, insulin, etc. Mammalian cell growth in bioreactors can be facilitated with microcarrier beads, allowing for increased yields. Bioreactors and microcarrier beads have been described in U.S. Pat. No. 5,073,491 to Familletti, U.S. Pat. No. 5,175,093 to Seifert, U.S. Pat. No. 5,654,197 to Jem, et al., U.S. Pat. No. 5,153,133 to Schwarz, et al., U.S. Pat. No. 4,906,577 to Armstrong, et al., U.S. Pat. No. 4,824,946 to Schwengers, et al., U.S. Pat. No. 4,189,534 to Levine, et al., and U.S. Pat. No. 6,150,581 to Jiang, et al. CULTISPHER (Percell Biolytica AB, Sweden) beads are constructed of porous gelatin, with random pore orientation and unpredictable interconnectivity of pores. However, these microcarrier beads do not provide the protection from an agitated fluid environment to allow aggressive oxygenation of the suspension and hence higher cell yields; it is recommended stirring be only active enough to prevent CULTISPHER beads from sedimenting. CYTODEX is another type of microcarrier beads, which also does not provide sufficient cell survival at a more agitated fluid environment, as CYTODEX beads are non-porous beads, which grow anchorage dependent cells upon their surface.
Despite the foregoing developments, there is a need in the art forimproved microcarrier beads capable of supporting cell growth in agitated conditions and providing larger areas for cell growth. Such beads would increase anchorage dependent cell yield in bioreactors as they would allow for increased suspension oxygenation due to stirring and sparging.
Three-dimensional (3D) scaffolds play important roles in scaffold guided tissue engineering because they provide critical functions as artificial extracellular matrices onto which cells can attach, grow, and form new tissues. Design and fabrication of tissue scaffolds are important issues in regenerative medicine, particularly for load bearing scaffolds in bone and cartilage tissue engineering application (see U.S. Pat. No. 5,900,207 to Danforth et al., U.S. Pat. No. 6,712,850 to Vyakamam et al., U.S. Pat. No. 6,730,252 to Teoh et al., and U.S. Pat. No. 6,645,412 to Friedman Jr.).
To design a 3D scaffold, one needs to address multiple biological, mechanical and geometrical design constraints and take into account scaffold external and internal geometry, porosity, pore size, pore interconnectivity, strength, transport properties, and microenvironment for cell and tissue ingrowth and healing (Hollister et al., 2002; Hutmacher, 2000; Sun and Lal, 2002). Advances in computer-aided tissue engineering and the use of biomimetic design made possible the introduction of biological and biophysical constraints into the scaffold design (Sun et al., 2003). However, thusly designed scaffolds often have intricate architectures that can only be fabricated through advanced manufacturing techniques. Most available scaffold fabrication methods, such as solvent casting, fiber bonding, phase separation, gas induced foaming, and salt leaching, either produce scaffolds with simple geometry or depend upon an indirect casting method for scaffold fabrication (Taboas et al., 2003; Yang et al., 2002). They are impractical for manufacturing scaffolds with complex structural architectures, and specifically complex internal architectures. To overcome this hurdle, solid freeform fabrication techniques, such as 3D printing, multi-phase jet solidification, and fused deposition modeling (FDM) have been widely adopted for scaffold fabrication (Koch et al., 1998; Wu et al., 1996; Zein et al., 2002). Among the reported techniques, FDM-based extruding deposition seems to be one of the most promising processes because of its versatility in using different scaffolding materials and the possibility of manufacturing scaffolds in a cell-friendly environment (Vozzi et al., 2002; Xiong et al., 2001). On the other hand, the ability to quantify the effect of the process on the morphology and the functional properties of the scaffolds is as important as the scaffold fabrication itself, because the biological and mechanical functions of the scaffold are in part dominated by the fabricated local micro-architecture of the scaffold. Micro-computed tomographic (micro-CT) imaging technology enables the characterization of the salient features of the scaffolds for tissue engineering applications. Recent reports have shown that micro-CT techniques are capable of characterizing micro-architectural and mechanical properties of tissue scaffolds (Lin et al., 2003), evaluating porous biomaterials (Muller et al., 1996), quantifying bone tissue morphologies and internal stress-strain behavior (Van et al., 1999), and conducting nondestructive evaluation for tissue properties (Muller and Ruegsegger, 1997). A recent study demonstrated the use of a precision extruding deposition (PED) process to fabricate poly-E-caprolactone (PCL) tissue scaffolds with designed micro-architecture, and then demonstrated the use of a micro-CT technique for evaluation and characterization of the morphologies and microstructures of the PED fabricated scaffolds. In contrast to the conventional FDM process that requires the use of precursor filaments, the PED process directly extrudes scaffolding materials from a granulated or pellet form without prior filament preparation.
An apparatus comprising a multi-nozzle biopolymer deposition system capable of extruding biopolymer solutions and living cells for freeform construction of three-dimensional tissue scaffolds is described in a PCT application Serial No. PCT/US2004/015316 filed on May 14, 2004 and U.S. patent application Ser. No. 10/540,968 incorporated herein in their entireties. The apparatus and the method do not describe depositing polycaprolactone (PCL).
The most common process of creating biocompatible structures is mold fabrication. While mold fabrication can create structures of desirable exterior shape, there is limited ability in controlling internal architecture. Secondary processes (such as salt fusion and phase manipulation) may be used to add porosity, but such processes often create random pores with unknown connectivity.
Currently, commercial applications of tissue engineering scaffolds are limited. Examples include films and gels such as those used in wound healing applications and rigid, porous filler materials such as those used to fill bone defects. Existing techniques need improvement when manufacturing of, tissue engineering scaffolds containing intact viable biomaterial is attempted.
Despite the foregoing developments, there is a need in the art for improved methods and apparatuses capable of producing porous structures with controllable pore sizes and pore interconnectivity, as well as the ability to incorporate biomaterial in a scaffold without affecting the biomaterial's viability.
All references cited herein are incorporated herein by reference in their entireties.